In teleosts，as in other vertebrates，the pituitary gonadotropic hormones，GtH I and GtHⅡ，play an important role in regulating gametogenesis. However，the mechanism of gonadotropinⅡβ-subunit gene transcriptional regulation has not been thoroughly understood yet. The present study was to get basic information of the possible cis-acting elements that involved in transcriptional regulation of the GtHⅡβ gene expression of blunt snout bream （Megalobrama amblycephala），and provide preconditions for further research on the molecular mechanism of these cis-acting elements in the GtHⅡβ gene transcriptional regulation. According to cDNA sequence information of blunt snout bream gonadotropinⅡβ-subunit （bGtHⅡβ）gene，the 5′ region of bGtHⅡβ was cloned by a simple method for cloning genomic DNA segments outside the boundaries of known sequences. In the first step of the method，a library of single-stranded flanking sequences is generated by linear amplification with one primer in the known region. Then a homooligomeric cytosine tail is added to each of the single-stranded fragments by a terminal transferase catalyzed reaction. The tailed fragments are amplified by PCR with a nested primer in the known region and a poly-guanine primer complementary to the cytosine tail in the unknown region. Finally，the different fragments are separated by cloning and characterized by sequencing.Sequence analysis reveals that the length of the 5 ′ flanking region of bGtHⅡβ gene is 1 354 bp and the region contains some potential transcription factors which may have important functions for the transcriptional regulation of the gene，such as ERE，ARE，PRE，GRE，LHX3，SF-1，Sp1，Pit-1，NF-Y，AP1 etc. And the promoter sequence is located on -  40 ～ 10 bp. Based on the above information，five partial deletion fragments as well as the full length of the 5 ′ flanking region were cloned from the genome by PCR and linked to a luciferase reporter gene. These partial fragments contained 811bp （-  771- +  40 bp），601 bp （-  561- +  40 bp），386 bp （-  346  - +  40 bp），239 bp （-  199  - +  40 bp） and 98 bp （-  58  - +  40 bp），respectively. It lays foundation for further research on mechanism of GtH Ⅱβ subunit gene transcriptional regulation.